EP3570032B1 - Procédé d'examen de la possibilité pour un sujet de souffrir d'un cancer du pancréas - Google Patents

Procédé d'examen de la possibilité pour un sujet de souffrir d'un cancer du pancréas Download PDF

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EP3570032B1
EP3570032B1 EP17891449.5A EP17891449A EP3570032B1 EP 3570032 B1 EP3570032 B1 EP 3570032B1 EP 17891449 A EP17891449 A EP 17891449A EP 3570032 B1 EP3570032 B1 EP 3570032B1
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gprc5c
exosome
concentration
present
pancreatic cancer
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EP3570032A4 (fr
EP3570032A1 (fr
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Takahiro Ochiya
Yusuke YOSHIOKA
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THEORIA SCIENCE Inc
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Theoria Science Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70525—ICAM molecules, e.g. CD50, CD54, CD102
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70546—Integrin superfamily, e.g. VLAs, leuCAM, GPIIb/GPIIIa, LPAM
    • G01N2333/70553—Integrin beta2-subunit-containing molecules, e.g. CD11, CD18
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705

Definitions

  • the present invention relates to a method of examining a possibility of a subject having pancreatic cancer, a method of distinguishing pancreatic cancer from a pancreatic disease other than pancreatic cancer, a method of monitoring a possibility of a subject having pancreatic cancer, a reagent for examining a possibility of a subject having pancreatic cancer, a reagent for distinguishing pancreatic cancer from a pancreatic disease other than pancreatic cancer, a reagent for monitoring a possibility of a subject having pancreatic cancer, a use of a pancreatic cancer marker, a pancreatic cancer diagnostic reagent, and a method of determining a biological sample.
  • An exosome is a granular vesicle present in a body fluid in a living body. It is known that, on an exosome surface, similar to a general cell surface, various membrane proteins are present. In addition, it is reported that the exosome is secreted from various cells, for example, cells in immune system or various cancer cells, and attention is paid to the fact that the exosome functions as an intermediate role for intercellular communication in a living body and is associated with physiological phenomenon, and association of the exosome with a disease such as cancer.
  • Non-Patent Document 1 a method of detecting colorectal cancer by measuring an amount of exosome expressing specific antigens (CD9, CD63, CD147) on an exosome surface (Patent Document 1), an increase of an amount of exosome in blood in accordance with progress of cancer in an ovarian cancer patient (Non-Patent Document 1), and the like have been reported.
  • Patent Document 2 an immunoassay method using a first antibody specifically binding to a first antigen present in the exosome and a second antibody specifically binding to a second antigen present in the exosome is known (Patent Document 2).
  • GPRC5C G protein-coupled receptor family C group 5 member C
  • GPRC5C is a 7-times transmembrane type G protein-coupled receptor, and it is reported that GPRC5C is highly expressed in the endocrine system such as adrenal gland, thyroid, and pancreas, and is used for diagnosis of endocrine diseases (Patent Document 3).
  • Non-Patent Document 1 Taylor., et al., Gynecologic Oncol, 100 (2008) pp!3-21
  • An object of the present invention is to provide a method and a reagent for examining a possibility of a subject having pancreatic cancer, a method and a reagent for distinguishing pancreatic cancer from a pancreatic disease other than pancreatic cancer, a method and a reagent for monitoring a possibility of a subject having pancreatic cancer, a use of pancreatic cancer marker, a pancreatic cancer diagnostic reagent, and a method for determining a biological sample.
  • pancreatic cancer can be distinguished from a pancreatic disease other than pancreatic cancer and a possibility of a subject having pancreatic cancer can be monitored by measuring GPRC5C present in an exosome in a specimen collected from a pancreatic cancer patient and a specimen collected from a pancreatic disease patient other than a pancreatic cancer patient, and thereby completing the present invention.
  • the invention is set out in the appended set of claims.
  • the present invention provides a method and a reagent for examining a possibility of a subject having pancreatic cancer, a method and a reagent for distinguishing pancreatic cancer from a pancreatic disease other than pancreatic cancer, a method and a reagent for monitoring the possibility of the subject having pancreatic cancer, and a use of pancreatic cancer marker, a pancreatic cancer diagnosis reagent, and a method for determining a biological sample.
  • a method for examining a possibility of a subject having pancreatic cancer 1.
  • a method for examining a possibility of a subject having pancreatic cancer of the present invention is a method of measuring a concentration of GPRC5C present in an exosome in a specimen collected from the subject.
  • An exosome in the present invention is a membrane vesicle covered with a lipid double membrane having a diameter of 30 to 100 nm secreted from an animal cell.
  • a specimen in the present invention is a specimen capable of measuring GPRC5C present in the exosome, and examples thereof include blood, urine, saliva, milk, nasal mucus, cerebrospinal fluid, and the like, and blood is preferable.
  • examples of the blood include whole blood, blood serum, plasma, and the like, and blood serum is preferable.
  • GPRC5C present in the exosome means any forms of GPRC5C such as GPRC5C contained in an exosome, GPRC5C present on a membrane surface of an exosome, and GPRC5C passing through a membrane of an exosome.
  • the concentration of GPRC5C present in an exosome includes not only an amount or molar amount per unit capacity but also signal intensity per unit capacity.
  • the method for examining a possibility of a subject having pancreatic cancer can be carried out by a method including the following steps.
  • Step (1) is a step of measuring a concentration of GPRC5C present in an exosome in the specimen collected.
  • the concentration of GPRC5C present in an exosome in the specimen collected can be measured by an immunoassay method, for example.
  • the immunoassay method is a method capable of measuring the concentration of GPRC5C present in an exosome in a specimen using antigen-antibody reaction. According to one embodiment, the immunoassay method is carried out by using an antibody binding to GPRC5C or a GPRC5C binding fragment thereof or by using an antibody binding to GPRC5C or a GPRC5C binding fragment thereof and an antibody binding to an exosome-specific antigen or an antigen binding fragment thereof.
  • the antibody binding to GPRC5C in step (1) is an antibody capable of measuring GPRC5C, and examples thereof include Anti-GPRC5C antibody-C-terminal (ab137482) (manufactured by Abcam Corporation), GPRC5C Antibody (N-14) (manufactured by Santa Cruz Biotechnology), and the like.
  • the exosome-specific antigen in step (2) include CD63, CD9, CD81, CD37, CD53, CD82, CD13, CD11, CD86, ICAM-1, Rab5, Annexin V, LAMP1, and the like.
  • the antibody binding to the exosome-specific antigen is an antibody capable of binding to an exosome-specific antigen.
  • Examples thereof include Purified Mouse Anti-Human CD63 (manufactured by Becton Dickinson Japan, clone: H5C6), Purified Mouse Anti-Human CD9 (manufactured by Becton Dickinson Japan, clone: M-L13), Purified Mouse Anti-Human CD81 (manufactured by Becton Dickinson Japan, clone: JS-81), Purified Mouse Anti-Human CD37 (manufactured by Becton Dickinson Japan, clone: M-B371), Purified Mouse Anti-Human CD53 (manufactured by Becton Dickinson Japan, clone: HI29), Anti-CD82 antibody [C33] (manufactured by Abcam Corporation), Purified Mouse Anti-Human CD13 (manufactured by Becton Dickinson Japan,
  • the fragment of the antibody binding to GPRC5C and the fragment of the antibody binding to an exosome-specific antigen in step (1) are fragments of antibodies capable of measuring GPRC5C and an exosome-specific antigen, respectively, and examples thereof include a fragment of an antibody obtained by removing an Fc portion such as Fab obtained by subjecting an antibody to papain treatment, F(ab') 2 obtained by pepsin treatment, and Fab' obtained by pepsin treatment-reduction treatment, a fragment of an antibody obtained by removing an Fc portion by gene engineering technique, and the like.
  • the concentration of GPRC5C present in an exosome in the specimen of step 1 can be measured by sandwich ELISA or ExoScreen method to be described later.
  • sandwich ELISA among the two kinds of antibodies used in measuring the concentration of GPRC5C present in the exosome, the antibody binding to an exosome-specific antigen or aan antigen bindingfragment thereof can be used as a solid-phase antibody, and the antibody binding to GPRC5C or a GPRC5C binding fragment thereof can be used as a labeled antibody.
  • the antibody binding to GPRC5C or a GPRC5 binding fragment thereof can be used as a solid-phase antibody, and the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof can be used as a labeled antibody.
  • Preparation of the solid-phase antibody and the labeled antibody can be carried out by a known method.
  • the measurement method of GPRC5C present in an exosome is carried out by using a method that is capable of measuring GPRC5C present in an exosome. According to one embodiment, a method such as an immunoassay method can be used.
  • an exosome including GPRC5C in a specimen binds to GPRC5C immobilized to a solid phase, subsequently, a labeled antibody binding to an exosome-specific antigen or an antige binding fragment thereof is added thereto, a complex of the antibody binding to GPRC5C or a GPRC5C binding fragment thereof, an exosome, and a labeled antibody binding to an exosome-specific antigen or an antigen binding fragment thereof is formed in a solid phase, the label in the formed complex is measured, and thereby GPRC5C present in the exosome can be measured.
  • the exosome in the specimen binds to an antibody binding to an exosome-specific antigen or an antige binding fragment thereof immobilized to a solid phase, subsequently, a labeled antibody binding to GPRC5C or a GPRC5C binding fragment thereof is added thereto, a complex of the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof, an exosome, and a labeled antibody binding to GPRC5C or a GPRC5C binding fragment thereof is formed in a solid phase, and the label in the formed complex is measured, and thereby GPRC5C present in the exosome can be measured.
  • various membranes such as microtiter plate, granular substances (beads) made of glass or synthetic resin, spherical substances (balls) made of glass or synthetic resin, latex, magnetic particles, nitrocellulose membrane, a test tube made of synthetic resin, and the like can be used.
  • an enzyme for example, an enzyme, a fluorescent substance, a light emission substance, a radioactive isotope, biotin, digoxigenin, polypeptide including a tag sequence, metal colloid particles, colored latex particles, and the like can be used.
  • alkali phosphatase for example, alkali phosphatase, peroxidase, galactosidase, glucuronidase, luciferase, and the like can be exemplified.
  • fluorescent substance for example, fluorescein isothiocyanate (FITC), rhodamine B isothiocyanate (RITC), and the like can be exemplified.
  • fluorescent substances for example, quantum dot ( Science, 281, 2016-2018, 1998 ), a phycobiliprotein such as phycoerythrin, and proteins emitting fluorescence such as GFP (Green fluorescent Protein), RFP (Redfluorescent Protein), YFP (Yellow fluorescent Protein), and BFP (Blue fluorescent Protein) are exemplified.
  • acridinium and the derivative a ruthenium complex compound, lophine, and the like are exemplified.
  • a ruthenium complex compound those shown in Clin. Chem. 37, 9, 1534-1539, 1991 , which electrochemically emit light along with an electron donor, are preferable.
  • radioactive isotope for example, 3 H, 14 C, 35 S, 32 P, 125 I, 131 I, and the like are exemplified.
  • FLAG peptide FLAG tag, Asp Tyr Lys Asp Asp Asp Asp Lys
  • polyhistidine His tag, His His His His His His
  • myc epitope peptide myc tag, Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
  • hemagglutinin epitope peptide HA tag, Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
  • step (1) in a case of measuring the concentration of GPRC5C present in the exosome in the specimen without disrupting the exosome in the specimen, as the antibody binding to GPRC5C or a GPRC5C binding fragment thereof, among the antibody binding to GPRC5C or a GPRC5C binding fragment, an antibody binding to an epitope of GPRC5C present on an exosome surface or a GPRC5C epitope binding fragment is preferably used.
  • an ExoScreen method is exemplified.
  • the ExoScreen method is a method applying AlphaLISA developed by PerkinElmer Corporation. In this method, using two kinds of antibodies of which epitopes are different, as one antibody, a biotinized antibody bonded to biotin, and as the other antibody, an antibody bonded to AlphaLISA acceptor bead are used to perform reaction with an analysis sample. After that, by adding a donor bead bonded to streptoavidine, the biotinized antibody and the donor bead bind to each other via streptoavidin, and the acceptor bead and the donor bead are adjacent to each other.
  • a singlet enzyme is generated from the donor bead, and when the singlet enzyme reaches the acceptor bead, light of 615 nm is emitted and can be detected as a signal.
  • the biotinized antibody in which the antibody binding to GPRC5C or a GPRC5C binding fragment thereof is bonded to biotin, and the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof bonded to AlphaLISA acceptor bead are reacted with GPRC5C present in the exosome in the specimen, and thereafter, by adding a donor bead bonded to streptoavidin, a complex of the acceptor bead, the exosome, and the donor bead is formed. Excitation light is irradiated on the donor bead of the complex to generate a singlet enzyme, and light generated by reaction of the generated singlet enzyme and the acceptor bead is measured.
  • biotinized antibody in which the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof is bonded to biotin, and the antibody binding to GPRC5C bonded to AlphaLISA acceptor bead or a GPRC5C binding fragment thereof are reacted with GPRC5C present in the exosome in the specimen, and thereafter, by adding a donor bead bonded to streptoavidin, a complex of the acceptor bead, the exosome, and the donor bead is formed. Excitation light is irradiated on the donor bead of the complex to generate a singlet enzyme, and light generated by reaction of the generated singlet enzyme and the acceptor bead is measured.
  • Step (2) is a step of comparing a concentration of GPRC5C present in the exosome in the specimen collected from the subject, obtained in step (1), with a concentration of GPRC5C present in an exosome in a specimen collected from a healthy person.
  • the concentration of GPRC5C present in the exosome in the specimen collected from the subject is higher than the concentration of GPRC5C present in the exosome in the specimen collected from the healthy person, it is determined that the possibility of the subject having pancreatic cancer is high, and in a case where the concentration of GPRC5C present in the exosome in the specimen collected from the subject is the same as or lower than the concentration of GPRC5C present in the exosome in the specimen collected from the healthy person, it is determined that the possibility of the subject having pancreatic cancer is low.
  • the concentration of GPRC5C present in the exosome in the specimen collected from the healthy person is a criteria value for determining the possibility of the subject having pancreatic cancer, and the concentration of GPRC5C present in the exosome in the specimen collected from the healthy person can be set by measuring concentrations of GPRC5C present in the exosome in the specimens collected from a plurality of healthy persons, and statistically analyzing.
  • the same kind of sample as that of the specimen collected from the subject is preferable.
  • the specimen collected from the subject is blood
  • blood is also preferably used as the specimen collected from the healthy person.
  • the method of comparing the concentration of GPRC5C present in the exosome in the specimen collected from the subject with the concentration of GPRC5C present in the exosome in the specimen collected from the healthy person is a known method (Steel method, t test, Wilcoxon test, and the like).
  • a ratio of the concentration of GPRC5C present in the exosome in the specimen collected from the subject with respect to the concentration of the exosome-specific antigen is compared with a ratio of the concentration of GPRC5C present in the exosome in the specimen collected from the healthy person with respect to the concentration of the exosome-specific antigen.
  • the exosome-specific antigen for example, the above-described exosome-specific antigen and the like are exemplified.
  • step (1) of measurement method 1 the concentration of GPRC5C present in the exosome in the specimen can be measured by using an exosome isolated from the specimen collected from the subject. Specifically, a method including the following steps is exemplified.
  • the method of isolating the exosome is a method capable of isolating the exosome in the specimen.
  • a method capable of isolating the exosome in the specimen for example, an ultracentrifugation method ( Trends in Molecular Medicine. 21, 533 (2015 )), a method of using exosome isolation kit EXO-Prep (manufactured by Cosmo Bio Co., Ltd), and the like are exemplified.
  • any method can be used as long as it is capable of measuring the concentration of GPRC5C present in the exosome.
  • an immunoassay method is used.
  • the measurement method of the concentration of GPRC5C present in the exosome by an immunoassay method the following aspects are exemplified.
  • a method of using an antibody binding to GPRC5C or a GPRC5C binding fragment thereof is a method of using an antibody binding to GPRC5C or a GPRC5C binding fragment thereof.
  • a method of using an antibody binding to GPRC5C or a GPRC5C binding fragment thereof and an antibody binding to an exosome-specific antigen or an antigen binding fragment thereof is a method of using an antibody binding to GPRC5C or a GPRC5C binding fragment thereof and an antibody binding to an exosome-specific antigen or an antigen binding fragment thereof.
  • the antibody binding to GPRC5C or a GPRC5C binding fragment thereof for example, the above-described antibody binding to GPRC5C or a GPRC5C binding fragment thereof and the like are exemplified.
  • the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof for example, the above-described antibody binding to an exosome-specific antigen or a antigen binding fragment thereof and the like are exemplified.
  • Western blotting for example, the method described in Monoclonal Antibodies-Principles and practices, Third edition, Academic Press (1996 ) and the like are exemplified.
  • the immunoassay method described in measurement method 1 for example, the ExoScreen method is exemplified.
  • the sandwich ELISA method and the like are exemplified.
  • the ExoScreen method for example, the above-described ExoScreen method and the like are exemplified.
  • an antibody binding to an epitope of GPRC5C present on an exosome surface or a GPRC5C epitope binding fragment thereof in a case of measurement without disrupting an isolated exosome, as the antibody binding to GPRC5C or a GPRC5C binding fragment thereof, among the antibody binding to GPRC5C or a GPRC5C binding fragment thereof, an antibody binding to an epitope of GPRC5C present on an exosome surface or a GPRC5C epitope binding fragment thereof.
  • the method of disrupting an isolated exosome for example, a method of using ultrasonic waves and the like are exemplified.
  • the sandwich ELISA method for example, the antibody binding to GPRC5C or a GPRC5C binding fragment thereof can be used as a solid-phase antibody, and the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof may be used as a labeled antibody, or the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof may be used as a solid-phase antibody, and the antibody binding to GPRC5C or a GPRC5C binding fragment thereof can be used as a labeled antibody.
  • solid phase and the label for example, known solid phase and label are respectively exemplified, and for example, the above-described solid phase and the above-described label are respectively used.
  • a method of preparing the solid phase-antibody and the labeled antibody for example, a known preparation method is exemplified.
  • Step (3A) is a step of determining a concentration of GPRC5C present in an exosome in the specimen from the concentration of GPRC5C measured in step (2A).
  • a concentration of GPRC5C present in an exosome in the specimen can be determined from the capacity of the specimen and the concentration of GPRC5C measured in step (3A).
  • Step (4A) is a step of comparing the concentration of GPRC5C present in an exosome in the specimen collected from the subject, determined in step (3A), with a concentration of GPRC5C present in an exosome in a specimen collected from a healthy person.
  • the concentration of GPRC5C present in the exosome in the specimen collected from the subject is higher than the concentration of GPRC5C present in the exosome in the specimen collected from the healthy person, it is determined that the possibility of the subject having pancreatic cancer is high, and in a case where the concentration of GPRC5C present in the exosome in the specimen collected from the subject is the same as or lower than the concentration of GPRC5C present in the exosome in the specimen collected from the healthy person, it is determined that the possibility of the subject having pancreatic cancer is low.
  • the concentration of GPRC5C present in the exosome in the specimen collected from the healthy person is a criteria value for determining the possibility of the subject having pancreatic cancer, and the concentration of GPRC5C present in an exosome in the specimen collected from the healthy person can be set by measuring concentrations of GPRC5C present in the exosome in the specimen collected from a plurality of healthy persons, and statistically analyzing.
  • the same kind of sample as that of the specimen of the subject is preferable.
  • the specimen of the subject is blood
  • blood is preferably used as the specimen of the healthy person.
  • the above-described method can be used. From the comparison, in a case where the concentration of GPRC5C present in the exosome in the specimen collected from the subject is shown to be higher than the concentration of GPRC5C present in the specimen collected from the healthy person, it is determined that the possibility of the subject having pancreatic cancer is high, and in a case where the concentration of GPRC5C present in the exosome in the specimen collected from the subject is shown to be the same as or lower than the concentration of GPRC5C present in the exosome in the specimen collected from the healthy person, it is determined that the possibility of the subject having pancreatic cancer is low.
  • a ratio of the concentration of GPRC5C present in the exosome in the specimen collected from the subject with respect to the concentration of the exosome-specific antigen is compared with a ratio of the concentration of GPRC5C present in the exosome in the specimen collected from the healthy person with respect to the concentration of the exosome-specific antigen.
  • the exosome-specific antigen for example, the above-described exosome-specific antigen and the like are exemplified.
  • pancreatic cancer a pancreatic disease other than pancreatic cancer in a pancreatic disease patient
  • the method of distinguishing pancreatic cancer from a pancreatic disease other than pancreatic cancer in a pancreatic disease patient of the present invention is a method of measuring a concentration of GPRC5C present in an exosome in a specimen collected from a pancreatic disease patient.
  • the specimen in the distinguishing method of the present invention is a specimen from which GPRC5C present in the exosome can be measured.
  • GPRC5C present in the exosome can be measured.
  • the above-described specimen and the like are exemplified.
  • GPRC5C present in the exosome may be present in any forms, for example, may be contained in the exosome, may be present on a membrane surface of the exosome, or may pass through a membrane of the exosome.
  • the concentration of GPRC5C present in the exosome includes not only a mass or a molar amount per unit capacity but also signal intensity per unit capacity.
  • the method of distinguishing pancreatic cancer from a pancreatic disease other than pancreatic cancer can be carried out by a method including the following steps.
  • any method can be used as long as the method is a method capable of measuring the concentration of GPRC5C present in the exosome.
  • an immunoassay method is used.
  • the measurement method of the concentration of GPRC5C present in the exosome by the immunoassay method the following aspects are exemplified.
  • a method of using an antibody binding to GPRC5C or a GPRC5C binding fragment thereof is a method of using an antibody binding to GPRC5C or a GPRC5C binding fragment thereof.
  • the antibody binding to GPRC5C or a GPRC5C binding fragment thereof in step (1B) is an antibody capable of measuring GPRC5C or a GPRC5C binding fragment thereof.
  • the above-described antibody or an antigen bindingfragment thereof and the like are exemplified.
  • step (1B) for example, the above-described antigen and the like are exemplified.
  • the antibody binding to the exosome-specific antigen or an antigen binding fragment thereof in step (1B) is an antibody capable of binding to an exosome-specific antigen or a antigen binding fragment thereof.
  • an antibody capable of binding to an exosome-specific antigen or a antigen binding fragment thereof for example, the above-described antibody or an antigen binding fragment thereof and the like are exemplified.
  • the concentration of GPRC5C present in the exosome can be measured by using the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof as a solid-phase antibody and using the antibody binding to GPRC5C or a GPRC5C binding fragment thereof as a labeled antibody.
  • the concentration of GPRC5C present in the exosome can be measured by using the antibody binding to GPRC5C or a GPRC5C binding fragment thereof as a solid-phase antibody and using the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof of the antibody as a labeled antibody.
  • Preparation of the solid-phase antibody and the labeled antibody can be carried out by a known method.
  • any method can be used as long as it is capable of measuring GPRC5C present in the exosome.
  • an immunoassay method such as the above-described sandwich ELISA and the ExoScreen method can be used.
  • step (1B) in a case of measuring the concentration of GPRC5C present in the exosome in the specimen without disrupting the exosome in the specimen, as an antibody binding to GPRC5C or a GPRC5C binding fragment thereof, among the above-described antibody binding to GPRC5C or a GPRC5C binding fragment thereof of, an antibody binding to an epitope of GPRC5C present on an exosome surface or a GPRC5C epitope binding fragment thereof is preferably used.
  • Step (2B) is a step of comparing the concentration of GPRC5C present in the exosome in the specimen collected from the pancreatic disease patient, obtained in step (1B), with the concentration of GPRC5C present in the exosome in the specimen collected from the patient having a pancreatic disease other than pancreatic cancer, and is a step of determining that the possibility of the pancreatic disease patient having pancreatic cancer is high in a case where the concentration of GPRC5C present in the exosome in the specimen collected from the pancreatic disease patient is higher than the concentration of GPRC5C in the specimen collected from the patient having a pancreatic disease other than pancreatic cancer, and determining that the possibility of the pancreatic disease patient having a pancreatic disease other than pancreatic cancer is high in a case where the concentration of GPRC5C present in the exosome in the specimen collected from the pancreatic disease patient is the same as or lower than the concentration of GPRC5C in the specimen collected from the patient having a pancreatic disease other
  • pancreatitis As the pancreatic disease other than pancreatic cancer, pancreatitis is exemplified. As the pancreatitis, chronic pancreatitis and acute pancreatitis are exemplified.
  • the concentration of GPRC5C present in the exosome in the specimen collected from the patient having a pancreatic disease other than pancreatic cancer is a criteria value for distinguishing whether the subject has pancreatic cancer or has a pancreatic disease other than pancreatic cancer, and the concentration of GPRC5C present in an exosome in the specimen collected from a patient having a pancreatic disease other than pancreatic cancer can be set by measuring concentrations of GPRC5C present in the exosome in the specimen collected from a plurality of patients having a pancreatic disease other than pancreatic cancer and statistically analyzing.
  • the same kind of sample as that of the specimen collected from the subject is preferable.
  • the specimen collected from the subject is blood
  • the specimen collected from the patient having a pancreatic disease other than pancreatic cancer blood is also preferably used.
  • the method of comparing the concentration of GPRC5C present in the exosome in the specimen collected from the subject with the concentration of GPRC5C present in the exosome in the specimen collected from the patient having a pancreatic disease other than pancreatic cancer is not particularly limited.
  • the above-described method can be used.
  • pancreatic disease patient as a criteria value for distinguishing pancreatic cancer from a pancreatic disease patient, instead of the concentration of GPRC5C present in the exosome in the specimen collected from a patient having pancreatic disease other than pancreatic cancer, a ratio of the concentration of GPRC5C present in the exosome in the specimen collected from the patient having pancreatic disease other than pancreatic cancer with respect to the exosome-specific antigen can be used.
  • a ratio of the concentration of GPRC5C present in the exosome in the specimen collected from the pancreatic disease patient with respect to the concentration of the exosome-specific antigen is compared with a ratio of the concentration of GPRC5C present in the exosome in the specimen collected from the patient having pancreatic disease other than pancreatic cancer with respect to the concentration of the exosome-specific antigen.
  • the exosome-specific antigen for example, the above-described exosome-specific antigen and the like are exemplified.
  • step (2B) of measurement method 3 the concentration of GPRC5C present in the exosome in the specimen can be measured by using an exosome isolated from the specimen collected from the subject. Specifically, a method including the following steps is exemplified.
  • step (1C) the method of isolating an exosome is a method capable of isolating an exosome in a specimen.
  • the above-described method and the like are exemplified.
  • any method can be used as long as it is capable of measuring a concentration of GPRC5C present in an exosome.
  • an immunoassay method is used.
  • the measurement method of the concentration of GPRC5C present in the exosome by the immunoassay method the following aspects are exemplified.
  • a method of using an antibody binding to GPRC5C or a GPRC5C binding fragment thereof is a method of using an antibody binding to GPRC5C or a GPRC5C binding fragment thereof.
  • a method of using an antibody binding to GPRC5C or a GPRC5C binding fragment tthereof and an antibody binding to an exosome-specific antigen or a an antigen binding fragment of the antibody is a method of using an antibody binding to GPRC5C or a GPRC5C binding fragment tthereof and an antibody binding to an exosome-specific antigen or a an antigen binding fragment of the antibody.
  • the antibody binding to GPRC5C or a GPRC5C binding fragment thereof for example, the above-described antibody binding to GPRC5C or a GPRC5C binding fragment thereof and the like are exemplified.
  • the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof for example, the above-described antibody binding to an exosome-specific antigen or an antige binding fragment theereof and the like are exemplified.
  • the immunoassay method described in measurement method 1 for example, the ExoScreen method and the like are exemplified, and in a case of measuring by disrupting the isolated exosome, the sandwich ELISA and the like are exemplified.
  • the ExoScreen method for example, the above-described ExoScreen method and the like are exemplified.
  • an antibody binding to GPRC5C or a GPRC5C binding fragment thereof among the above-described antibody binding to GPRC5C or a GPRC5C binding fragment thereof, an antibody binding to an epitope of GPRC5C present on an exosome surface or a GPRC5C eptope binding fragment is used.
  • the antibody binding to GPRC5C or a GPRC5C binding fragment thereof may be used as a solid-phase antibody and the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof may be used as a labeled antibody, and the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof may be used as a solid-phase antibody and the antibody binding to GPRC5C or a GPRC5C binding fragment thereof may be used as a labeled antibody.
  • the solid phase or the label for example, known solid phase and label are respectively exemplified.
  • the above-described solid phase and the label are respectively used.
  • a method of preparing the solid-phase antibody and the labeled antibody for example, a known preparation method is exemplified.
  • Step (3C) is a step of determining a concentration of GPRC5C present in an exosome in the specimen from the concentration of GPRC5C measured in step (2C).
  • a concentration of GPRC5C present in an exosome of the specimen can be determined from the capacity of the specimen and the concentration of GPRC5C measured in step (3C).
  • Step (4C) is a step of comparing a concentration of GPRC5C present in the exosome in the specimen collected from the pancreatic disease patient, determined in step (3C), with a concentration of GPRC5C present in an exosome in a specimen collected from a patient having a pancreatic disease other than pancreatic cancer.
  • the concentration of GPRC5C present in the exosome in the specimen collected from the pancreatic disease patient is higher than the concentration of GPRC5C present in the exosome in the specimen collected from the patient having a pancreatic disease other than pancreatic cancer, it is determined that the possibility of the pancreatic disease patient having pancreatic cancer is high, and in a case where the concentration of GPRC5C present in the exosome in the specimen collected from the patient having a pancreatic disease is the same as or lower than the concentration of GPRC5C present in the exosome in the specimen collected from the patient having a pancreatic disease other than pancreatic cancer, it is determined that the possibility of the pancreatic disease patient having a pancreatic disease other than pancreatic cancer is high.
  • the concentration of GPRC5C present in the exosome in the specimen collected from the patient having a pancreatic disease other than pancreatic cancer is a criteria value for distinguishing whether the pancreatic disease patient has pancreatic cancer or has a pancreatic disease other than pancreatic cancer, and the concentration of GPRC5C present in the exosome in the specimen collected from the patient having a pancreatic disease other than pancreatic cancer can be set by measuring concentrations of GPRC5C present in the exosome in the specimens collected from a plurality of patients having a pancreatic disease other than pancreatic cancer and statistically analyzing.
  • the specimen of the patient having a pancreatic disease other than pancreatic cancer used at a time of setting the criteria value is preferably the same kind of sample as that of the specimen of the pancreatic disease patient which is a subject.
  • the specimen of the pancreatic disease patient which is a subject is blood
  • blood is also preferably used as the specimen of the patient having a pancreatic disease other than pancreatic cancer.
  • the above-described method can be used.
  • pancreatic disease patient as a criteria value for distinguishing pancreatic cancer from a patient having a pancreatic disease other than pancreatic cancer, instead of the concentration of GPRC5C present in the exosome in the specimen collected from a patient having pancreatic disease other than pancreatic cancer, a ratio of the concentration of GPRC5C present in the exosome in the specimen collected from the patient having pancreatic disease other than pancreatic cancer with respect to the concentration of the exosome-specific antigen can be used.
  • a ratio of the concentration of GPRC5C present in the exosome in the specimen collected from the pancreatic disease patient with respect to the concentration of the exosome-specific antigen is compared with a ratio of the concentration of GPRC5C present in the exosome in the specimen collected from the patient having pancreatic disease other than pancreatic cancer with respect to the concentration of the exosome-specific antigen.
  • the exosome-specific antigen for example, the above-described exosome-specific antigen and the like are exemplified.
  • the method of monitoring a possibility of a subject having pancreatic cancer of the present invention is a method of measuring a concentration of GPRC5C present in an exosome in a specimen sequentially collected from the subject.
  • the specimen in the present invention is not particularly limited as long as it is capable of measuring GPRC5C present in an exosome.
  • the above-described specimen and the like are exemplified.
  • GPRC5C present in the exosome may be present in any forms, for example, may be contained in an exosome, may be present on a membrane surface of an exosome, or may pass through a membrane of an exosome.
  • the concentration of GPRC5C present in an exosome includes not only an amount or molar amount per unit capacity but also signal intensity per unit capacity.
  • the method of monitoring a possibility of a subject having pancreatic cancer can be carried out by a method including the following steps.
  • a method capable of measuring a concentration of GPRC5C present in an exosome As the method of measuring the concentration of GPRC5C present in an exosome in step (1D), a method capable of measuring a concentration of GPRC5C present in an exosome. According to one embodiment, a method such as an immunoassay method is used. As the measurement method of the concentration of GPRC5C present in an exosome by an immunoassay method, the following aspects are exemplified.
  • a method of using an antibody binding to GPRC5C or a GPRC5C binding fragment thereof is a method of using an antibody binding to GPRC5C or a GPRC5C binding fragment thereof.
  • a method of using an antibody binding to GPRC5C or a GPRC5C binding fragment thereof and an antibody binding to an exosome-specific antigen or an antigen binding fragment thereof is a method of using an antibody binding to GPRC5C or a GPRC5C binding fragment thereof and an antibody binding to an exosome-specific antigen or an antigen binding fragment thereof.
  • the antibody binding to GPRC5C or a GPRC5C binding fragment thereof of the antibody in step (1D) is an antibody capable of measuring GPRC5C or a GPRC5C binding fragment thereof.
  • the above-described antibody or an antigen binding fragment thereof and the like are exemplified.
  • step (1D) for example, the above-described antigen and the like are exemplified.
  • the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof in step (1D) is an antibody capable of binding to an exosome-specific antigen or an antigen binding fragment thereof.
  • the concentration of GPRC5C present in an exosome can be measured by using the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof as a solid-phase antibody and using the antibody binding to GPRC5C or a GPRC5C binding fragment thereof as a labeled antibody.
  • the concentration of GPRC5C present in an exosome can be measured by using the antibody binding to GPRC5C or a GPRC5C binding fragment thereof as a solid-phase antibody and using the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof as a labeled antibody.
  • Preparation of the solid-phase antibody and the labeled antibody is not particularly limited, and can be carried out by a known method.
  • the measurement method of GPRC5C present in an exosome can be carried out by using any method as long as it is capable of measuring GPRC5C present in an exosome.
  • an immunoassay method such as the above-described sandwich ELISA and ExoScreen method and the like can be used.
  • step (1D) in a case of measuring the concentration of GPRC5C present in the exosome in the specimen without disrupting the exosome in the specimen, as the antibody binding to GPRC5C or a GPRC5C binding fragment thereof the above-described antibody binding to GPRC5C or a GPRC5C binding fragment thereof, an antibody binding to an epitope of GPRC5C present on an exosome surface or a GPRC5C epitope binding fragment thereof is preferably used.
  • Step (2D) is a method of comparing the concentration of GPRC5C present in the exosome in the specimen collected from the subject, obtained in step (1D), with the concentration of GPRC5C present in the exosome in the specimen collected from a healthy person.
  • the possibility of the subject having pancreatic cancer is high in a case where the concentration of GPRC5C present in the exosome in the specimen collected from the subject is higher than a concentration of GPRC5C present in an exosome in a specimen collected from a healthy person, and it is determined that the possibility of the subject having pancreatic cancer is low in a case where the concentration of GPRC5C present in the exosome in the specimen collected from the subject is the same as or lower than the concentration of GPRC5C present in the exosome in the specimen collected from the healthy person.
  • the concentration of GPRC5C present in the exosome in the specimen collected from the healthy person is a criteria value for determining the possibility of the subject having pancreatic cancer, and the concentration of GPRC5C present in an exosome in a specimen collected from the healthy person can be set by measuring concentrations of GPRC5C present in the exosome in the specimens collected from a plurality of healthy persons and statistically analyzing.
  • the same kind of sample as that of the specimen collected from the subject is preferable.
  • the specimen collected from the subject is blood
  • blood is also preferably used as the specimen collected from the healthy person.
  • the method of comparing the concentration of GPRC5C present in the exosome in the specimen sequentially collected from the subject with the concentration of GPRC5C present in the exosome in the specimen collected from the healthy person is not particularly limited.
  • the above-described method and the like can be used.
  • a ratio of the concentration of GPRC5C present in the exosome in the specimen collected from the healthy person with respect to the concentration of the exosome-specific antigen can be used.
  • the ratio of the concentration of GPRC5C present in the exosome in the specimen collected from the subject with respect to the concentration of the exosome-specific antigen is compared with the ratio of the concentration of GPRC5C present in the exosome in the specimen collected from the healthy person with respect to the concentration of the exosome-specific antigen.
  • the exosome-specific antigen for example, the above-described exosome-specific antigen and the like are exemplified.
  • step (1D) of measurement method 5 the concentration of GPRC5C present in the exosome in the specimen can be measured by using an exosome isolated from a specimen collected from the subject. Specifically, a method including the following steps is exemplified.
  • step (1E) the method of isolating an exosome is a method capable of isolating an exosome in a specimen.
  • the above-described method and the like are exemplified.
  • any method can be used as long as it is capable of measuring a concentration of GPRC5C present in an exosome.
  • a method such as an immunoassay method is used.
  • the measurement method of the concentration of GPRC5C present in the exosome by the immunoassay method the following aspects are exemplified.
  • a method of using an antibody binding to GPRC5C or a GPRC5C binding fragment thereof is a method of using an antibody binding to GPRC5C or a GPRC5C binding fragment thereof.
  • a method of using an antibody binding to GPRC5C or a GPRC5C binding fragment thereof and an antibody binding to an exosome-specific antigen or a an antigen binding fragment thereof is a method of using an antibody binding to GPRC5C or a GPRC5C binding fragment thereof and an antibody binding to an exosome-specific antigen or a an antigen binding fragment thereof.
  • the antibody binding to GPRC5C or a GPRC5C binding fragment thereof for example, the above-described antibody binding to GPRC5C or a GPRC5C binding fragment thereof and the like are exemplified.
  • the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof for example, the above-described antibody binding to an exosome-specific antigen or an antigen bindingfragment thereof and the like are exemplified.
  • the immunoassay method described in measurement method 1 for example, an ExoScreen method and the like are exemplified, and in a case of measuring by disrupting the isolated exosome, a sandwich ELISA method and the like are exemplified.
  • the ExoScreen method for example, the above-described ExoScreen method and the like are exemplified.
  • an antibody binding to an epitope of GPRC5C present on an exosome surface or a GPRC5C epitope binding fragment is used.
  • the antibody binding to GPRC5C or a GPRC5C binding fragment thereof may be used as a solid-phase antibody and the antibody binding to an exosome-specific antigen or an antige binding fragment thereof may be used as a labeled antibody, and the antibody binding to an exosome-specific antigen or a fragment of the antibody may be used as a solid-phase antibody and the antibody binding to GPRC5C or a GPRC5C binding fragment thereof may be used as a labeled antibody.
  • the solid phase and the label for example, known solid phase and label are respectively exemplified.
  • the above-described solid phase and label are respectively used.
  • a method of preparing the solid-phase antibody and the labeled antibody for example, a known preparation method is exemplified.
  • Step (3E) is a step of determining a concentration of GPRC5C in an exosome in the specimen from the concentration of GPRC5C measured in step (2E).
  • the concentration of GPRC5C present in the exosome in the specimen can be determined from the capacity of the specimen and the concentration of GPRC5C measured in step (2E).
  • Step (4E) is a step of comparing the concentration of GPRC5C present in the exosome in the specimen collected from the subject, determined in step (3E), with the concentration of GPRC5C present in the exosome in the specimen collected from a healthy person.
  • the possibility of the subject having pancreatic cancer is high in a case where the concentration of GPRC5C present in the exosome in the specimen collected from the subject is maintained at a concentration higher than a concentration of GPRC5C present in an exosome in a specimen collected from a healthy person, and it is determined that the possibility of the subject having pancreatic cancer is low in a case where the concentration of GPRC5C present in the exosome in the specimen collected from the subject is maintained at a concentration the same as or lower than the concentration of GPRC5C present in the exosome in the specimen collected from the healthy person.
  • the concentration of GRPC5C present in the exosome in the specimen collected from the healthy person is a criteria value for determining the possibility of the subject having pancreatic cancer, and the concentration of GPRC5C present in the exosome in the specimen collected from the healthy person can be set by measuring concentrations of GPRC5C present in the exosome in the specimen collected from a plurality of healthy persons and statistically analyzing.
  • the same kind of sample as that of the specimen of the subject is preferable.
  • the subject's specimen is blood
  • blood is also preferably used as the specimen of the healthy person.
  • the above-described method can be used. From the comparison, in a case where the concentration of GPRC5C present in the exosome in the specimen collected from the subject is maintained at a concentration higher than the concentration of GPRC5C present in the exosome in the specimen collected from the healthy person, it is determined that the possibility of the subject having pancreatic cancer is high, and in a case where the where the concentration of GPRC5C present in the exosome in the specimen collected from the subject is maintained at a concentration the same as or lower than the concentration of GPRC5C present in the exosome in the subject collected from the healthy person, it is determined that the possibility of the subject having pancreatic cancer is low.
  • a ratio of the concentration of GPRC5C present in the exosome in the specimen collected from the healthy person with respect to the concentration of the exosome-specific antigen can be used instead of the concentration of GPRC5C present in the exosome in the specimen collected from the healthy person.
  • the ratio of the concentration of GPRC5C present in the exosome in the specimen collected from the subject with respect to the concentration of the exosome-specific antigen is compared with the ratio of the concentration of GPRC5C present in the exosome in the specimen collected from the healthy person with respect to the concentration of the exosome-specific antigen.
  • the exosome-specific antigen for example, the above-described exosome-specific antigen and the like are exemplified.
  • a pancreatic cancer patient's state such as therapeutic effects after performing operation and decrease in cancer sites can be monitored by measuring a concentration of GPRC5C present in an exosome in a specimen collected from a pancreatic cancer patient from a time before operation to a time after operation.
  • recurrence of pancreatic cancer can be predicted by measuring a concentration of GPRC5C present in an exosome in a specimen collected from a pancreatic cancer patient during treatment.
  • a possibility of a subject having pancreatic cancer can be monitored by measuring a concentration of GPRC5C present in an exosome in a specimen collected from the subject.
  • the reagent for examining the possibility of the subject having pancreatic cancer of the present invention is a reagent that contains a reagent for measuring GPRC5C present in an exosome, and can be used in the method of examining the possibility of the subject having pancreatic cancer of the present invention.
  • the reagent for examining the possibility of the subject having pancreatic cancer of the present invention may further contain a criteria table that describes criteria in which, in a case where a concentration of GPRC5C present in an exosome in a specimen collected from the subject is higher than a concentration of GPRC5C present in an exosome in a specimen collected from a healthy person, the possibility of the subject having pancreatic cancer is high, and in a case where the concentration of GPRC5C present in the exosome in the specimen collected from the subject is the same as or lower than the concentration of GPRC5C present in the exosome in the specimen collected from the healthy person, the possibility of the subject having pancreatic cancer is low.
  • the reagent for measuring GPRC5C present in an exosome is a reagent used in the above-described method of measuring GPRC5C present in an exosome.
  • the reagent for measuring GPRC5C present in an exosome is capable of measuring GPRC5C present in an exosome in a specimen.
  • This reagent is an immunoassay reagent..
  • a reagent including an antibody binding to GPRC5C or a GPRC5C binding fragment thereof.
  • a reagent including an antibody binding to GPRC5C or a GPRC5C binding fragment thereof to which a label is bonded.
  • a reagent including an antibody binding to GPRC5C or a GPRC5C binding fragment thereof and an antibody binding to an exosome-specific antigen or a antigen binding fragment thereof.
  • a reagent including an antibody binding to GPRC5C or a GPRC5C binding fragment immobilized to a solid phase, and an antibody binding to an exosome-specific antigen or an antigen binding fragment to which a label is bonded.
  • a reagent including an antibody binding to an exosome-specific antigen or an antigen binding fragment thereof immobilized to a solid phase, and an antibody binding to GPRC5C or a GPRC5C binding fragment thereof to which a label is bonded.
  • a reagent including a first antibody binding to GPRC5C or a GPRC5C binding fragment thereof, immobilized to a solid phase, and a second antibody binding to GPRC5C or a GPRC5C binding fragment thereof to which a label is bonded.
  • the above-described antibody binding to GPRC5C or a GPRC5C binding fragment thereof, and the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof are respectively exemplified.
  • the label in the antibody binding to GPRC5C or a GPRC5C binding fragment thereof to which a label is bonded and the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof to which a label is bonded, for example, the above-described label and the like are exemplified.
  • Preparation of the antibody binding to GPRC5C or a GPRC5C binding fragment thereof to which a label is bonded, and the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof to which a label is bonded can be carried out by a known preparation method of a labeled antibody, for example.
  • the above-described solid phase and the like are exemplified, for example. Immobilization of the antibody binding to GPRC5C or a GPRC5C binding fragment thereof to a solid phase, and immobilization of the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof to a solid phase can be carried out by a known method, for example.
  • an epitope to which the first antibody binding to GPRC5C or a GPRC5C epitope binding fragment binds and an epitope to which the second antibody binding to GPRC5C or a GPRC5C binding fragment thereof binds may be different or the same, but being different is preferable.
  • the measurement reagents 1 to 6 may further contain an exosome isolation reagent.
  • the exosome isolation reagent is a reagent capable of isolating an exosome from a specimen.
  • the above-described exosome isolation kit and the like are exemplified.
  • a reagent for distinguishing pancreatic cancer from a pancreatic disease other than pancreatic cancer in a pancreatic disease patient of the present invention is a reagent containing a reagent for measuring GPRC5C present in an exosome, and can be used in the method of distinguishing pancreatic cancer from a pancreatic disease other than pancreatic cancer in a pancreatic disease patient of the present invention.
  • the reagent for distinguishing pancreatic cancer from a pancreatic disease other than pancreatic cancer in a pancreatic cancer patient of the present invention may further contain a criteria table in which, in a case where a concentration of GPRC5C present in an exosome in a specimen collected from a pancreatic disease patient is higher than a concentration of GPRC5C present in an exosome in a specimen collected from a patient having a pancreatic disease other than pancreatic cancer, the possibility of pancreatic disease patient having pancreatic cancer is high, and in a case where the concentration of GPRC5C present in the exosome in the specimen collected from the patient having a pancreatic disease is the same as or lower than the concentration of GPRC5C present in the exosome in the specimen collected from the patient having a pancreatic disease other than pancreatic cancer, the possibility of pancreatic disease patient having a pancreatic disease other than pancreatic cancer is high.
  • the reagent for measuring GPRC5C present in an exosome is a reagent used in the above-described method of measuring GPRC5C present in an exosome.
  • the reagent for measuring GPRC5C present in the exosome is capable of measuring GPRC5C present in an exosome in a specimen.
  • the reagent is an immunoassay reagent.
  • a reagent including an antibody binding to GPRC5C or a GPRC5C binding fragment thereof.
  • a reagent including an antibody binding to GPRC5C or a GPRC5C binding fragment thereof to which a label is bonded.
  • a reagent including an antibody binding to GPRC5C or a GPRC5C binding fragment thereof, and an antibody binding to an exosome-specific antigen or an antigen binding fragment thereof.
  • a reagent including an antibody binding to GPRC5C or a GPRC5C binding fragment thereof to a solid phase, and an antibody binding to an exosome-specific antigen or an antigen binding fragment to which a label is bonded.
  • a reagent including an antibody binding to an exosome-specific antigen or an antigen binding fragment thereof of the antibody immobilized to a solid phase, and an antibody binding to GPRC5C or a GPRC5C binding fragment thereof to which a label is bonded.
  • a reagent including a first antibody binding to GPRC5C or a GPRC5C binding fragment thereof immobilized to a solid phase, and a second antibody binding to GPRC5C and a GPRC5C binding fragment thereof to which a label is bonded.
  • the above-described antibody binding to GPRC5C or a GPRC5C binding fragment thereof, and the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof are respectively exemplified.
  • the above-described label and the like are exemplified, for example.
  • Preparation of the antibody binding to GPRC5C or a GPRC5C binding fragment thereof to which a label is bonded, and the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof to which a label is bonded can be carried out by a known preparation method of a labeled antibody, for example.
  • the above-described solid phase and the like are exemplified, for example. Immobilization of the antibody binding to GPRC5C or a GPRC5C binding fragment thereof to a solid phase and immobilization of the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof to a solid phase can be carried out by a known method, for example.
  • an epitope to which the first antibody binding to GPRC5C or a GPRC5C binding fragment thereof binds, and an epitope to which the second antibody binding to GPRC5C or a GPRC5C binding fragment thereof binds may be different or the same, but being different is preferable.
  • the measurement reagents 7 to 12 may further contain an exosome isolation reagent.
  • the exosome isolation reagent is not particularly limited as long as it is a reagent capable of isolating an exosome from a specimen.
  • the above-described exosome isolation reagent and the like are exemplified.
  • the reagent for monitoring the possibility of the subject having pancreatic cancer of the present invention is a reagent containing a reagent for measuring GPRC5C present in an exosome, and can be used in the method of monitoring the possibility of the subject having pancreatic cancer of the present invention.
  • the reagent for monitoring the possibility of the subject having pancreatic cancer may further contain a criteria table that describes criteria in which, in a case where a concentration of GPRC5C present in an exosome in a specimen sequentially collected from the subject is maintained at a concentration higher than a concentration of GPRC5C present in an exosome in a specimen collected from a healthy person, the possibility of the subject having pancreatic cancer is high, and in a case where the concentration of GPRC5C present in the exosome in the specimen sequentially collected from the subject is maintained at a concentration the same as or lower than the concentration of GPRC5C present in the exosome in the specimen collected from the healthy person, the possibility of the subject having pancreatic cancer is low.
  • the reagent for measuring GPRC5C present in an exosome is a reagent used in the above-described method of measuring GPRC5C present in the exosome.
  • the reagent for measuring GPRC5C present in the exosome is a reagent capable of measuring GPRC5C present in an exosome in a specimen. This reagent is an an immunoassay reagent..
  • a reagent including an antibody binding to GPRC5C or a GPRC5C binding fragment thereof.
  • a reagent including an antibody binding to GPRC5C or a GPRC5C binding fragment thereof to which a label is bonded.
  • a reagent including an antibody binding to GPRC5C or a GPRC5C binding fragment thereof, and an antibody binding to an exosome-specific antigen or an antigen binding fragment.
  • a reagent including an antibody binding to GPRC5C or a GPRC5C binding fragment thereof immobilized to a solid phase, and an antibody binding to an exosome-specific antigen or an antigen binding fragment thereof to which a label is bonded.
  • a reagent including an antibody binding to an exosome-specific antigen or an antigen binding fragment thereof immobilized to a solid phase, and an antibody binding to GPRC5C or a GPRC5C binding fragment thereof to which a label is bonded.
  • a reagent including a first antibody binding to GPRC5C or a GPRC5C binding fragment thereof immobilized to a solid phase, and a second antibody binding to GPRC5C or a GPRC5C binding fragment thereof to which a label is bonded.
  • the antibody binding to GPRC5C or a GPRC5C binding fragment thereof, and the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof are respectively exemplified.
  • the label in the antibody binding to GPRC5C or a GPRC5C binding fragment thereof to which a label is bonded and the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof to which a label is bonded, for example, the above-described label and the like are exemplified.
  • Preparation of the antibody binding to GPRC5C or a GPRC5C binding fragment thereof to which a label is bonded, and the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof to which a label is bonded can be carried out by a known preparation method of a labeled antibody, for example.
  • the solid phase in the antibody binding to GPRC5C or a GPRC5C binding fragment thereof immobilized to a solid phase and the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof immobilized to a solid phase, for example, the above-described solid phase and the like are exemplified.
  • Immobilization of the antibody binding to GPRC5C or a GPRC5C binding fragment thereof to a solid phase and immobilization of the antibody binding to an exosome-specific antigen or an antigen binding fragment thereof to a solid phase can be carried out by a known method, for example.
  • an epitope to which the first antibody binding to GPRC5C or a GPRC5C binding fragment thereof, and an epitope to which the second antibody binding to GPRC5C or a GPRC5C binding fragment thereof may be different or the same, but being different is preferable.
  • the measurement reagents 13 to 18 may further contain an exosome isolation reagent.
  • the exosome isolation reagent is a reagent capable of isolating an exosome from a specimen.
  • the above-described exosome isolation kit and the like are exemplified.
  • a pancreatic cancer marker used according to the present invention includes GPRC5C or a GPRC5C gene.
  • the accession number of GenBank of human GPRC5C is NP_071319.2; NP_061123.3.
  • the accession number of GenBank of mRNA of human GPRC5C is NM_022036.2; NM_018653.3.
  • a pancreatic cancer diagnosis reagent of the present invention includes a specific binding substance to GPRC5C, a primer set capable of amplifying a GPRC5C gene, or a probe specifically hybridized to mRNA of a GPRC5C gene.
  • an antibody binding to GPRC5C an antibody binding to GPRC5C, a GPRC5C binding fragment thereof, an aptamer binding to GPRC5C, and the like are exemplified.
  • the antibody or an antigen binding fragment thereof the above-described ones are exemplified.
  • the aptamer is a substance having a specific binding ability to a target substance.
  • a nucleic acid aptamer, a peptide aptamer, and the like are exemplified.
  • a nucleic acid aptamer having a specific binding ability to a target substance can be selected by a method of systematic evolution of ligand by exponential enrichment (SELEX) and the like, for example.
  • a peptide aptamer having a specific binding ability to a target peptide can be selected by a Two-hybrid method using yeast and the like, for example.
  • the primer set capable of amplifying a GPRC5C gene is not particularly limited as long as it is capable of amplifying mRNA of the gene by an RT-PCR method and the like.
  • mRNA of the gene has a base sequence described in the GenBank database specified by the above-described accession number. Since there is a case where a splicing variant and the like are present in mRNA, mRNA of the gene is not limited to the one specified by the above-described accession number.
  • mRNA of a GPRC5C gene in a sample can be detected by performing RT-PCR using this primer set using a tissue of the subject and the like as the sample.
  • the probe is specifically hybridized to mRNA of a GPRC5C gene.
  • the probe may be immobilized to a carrier to constitute a DNA microarray and the like. For example, whether or not the subject has pancreatic cancer can be determined by contacting mRNA extracted from the tissue of the subject with the DNA microarray and detecting mRNA of the GPRC5C gene hybridized to the probe.
  • the carrier for example, the above-described carrier and the like are exemplified.
  • a method of determining a biological sample of the present invention includes a step of measuring an amount of GPRC5C in a biological sample and a step of determining that the biological sample is collected from a pancreatic cancer patient in a case where the measured amount of GPRC5C is larger compared to an amount of GPRC5C in a biological sample of a healthy person or a patient having a pancreatic disease other than pancreatic cancer, preferably pancreatitis.
  • pancreatic cancer can be specifically detected.
  • the biological sample may be plasma, blood serum, or an exosome purified from plasma or blood serum, as described above.
  • the step of measuring GPRC5C in the biological sample can be carried out by detection using sandwich ELISA, Western blotting, and reversed-phase protein array, and by immunohistochemical staining, using the above-described specific binding substance.
  • the measured GPRC5C may be converted into a quantitative value based on a calibration curve prepared by using serially diluted GPRC5C with a known concentration as a standard
  • the biological sample in a case where the amount of GPRC5C in the biological sample is larger than the amount of GPRC5C in a control sample, it is determined that the biological sample is collected from a pancreatic cancer patient.
  • a biological sample collected from a healthy person or a patient having a pancreatic disease other than pancreatic cancer is exemplified.
  • a criteria value is set based on the amount of GPRC5C present in the biological sample collected from a healthy person in advance, and the amount of GPRC5C is larger than the criteria value, it may be determined that the biological sample is collected from a pancreatic cancer patient.
  • the determination method of the present invention may include a step of measuring an expression amount of a GPRC5C gene in a biological sample and a step of determining that the biological sample is collected from a pancreatic cancer patient in a case where the measured expression amount of the gene is larger than that of a control.
  • the biological sample may be a tissue of the subject and the like.
  • the step of measuring the expression amount of a GPRC5C gene in the biological sample can be carried out by RT-PCR, quantitative RT-PCR using the above-described primer set, and the like.
  • the expression amount of a GPRC5C gene may be measured by DNA microarray analysis, Northern blotting using the above-described probe, and the like.
  • the measured expression amount of the GPRC5C gene may be converted into a quantitative value based on a calibration curve prepared by using a serially diluted fragment of each gene with a known concentration and the like as a standard.
  • the biological sample in a case where the expression amount of the GPRC5C gene in the biological sample is larger than the expression amount of the gene in the control sample, it is determined that the biological sample is collected from a pancreatic cancer patient.
  • the control for example, a biological sample collected from a healthy person or a patient having a pancreatic disease other than pancreatic cancer and the like are exemplified.
  • a criteria value is set based on the expression amount of the GPRC5C gene in the biological sample collected from a healthy person in advance, and the expression amount of the gene is larger than the criteria value, it may be determined that the biological sample is collected from a pancreatic cancer patient.
  • Blood serum (4 specimens) collected from healthy persons, blood serum (4 specimens) collected from pancreatitis patients, and blood serum (4 specimens: two kinds of stage IIA, one kind of stage IIB, and one kind of stage III) collected from pancreatic cancer patients were subjected to ultracentrifugation for 70 minutes in a swing rotor at 100,000 ⁇ g or greater (4°C), the supernatant was discarded, PBS [10 mmol/L phosphoric buffer solution (pH 7.2) containing 0.15 mol/L sodium chloride] was added thereto and precipitated, and the precipitated fraction was used as an exosome fraction. The exosome fraction was dissolved in PBS (total amount 100 ⁇ L).
  • exosome fraction solution equivalent to 100 ⁇ L blood serum per well was added and mixed with 4 ⁇ sample buffer (0.125 mol/L Tris buffer solution containing 4% dodecyl sodium sulfate, 10% sucrose, 0.01% bromophenol blue, 10% 2-mercaptoethanol [pH 6.8]) of 1/3 amount of the solution, kept warm at 95°C for 5 minutes, and then ice-cooled.
  • the treatment solution was applied to a well of 4-15% Mini-PROTEAN TGX gel (manufactured by Bio-Rad), and SDS-PAGE was performed.
  • the gel after SDS-PAGE was transferred to a PVDF membrane, and the membrane was subjected to blocking at room temperature (25°C) for 1 hour by using a Nacalai Blocking One solution [manufactured by Nacalai Tesque, Inc.]. Subsequently, primary antibody reaction was carried out at room temperature (25°C) for 1 hour (Anti-GPRC5C antibody C-terminal Rabbit polyclonal [manufactured by Abcam Corporation] 1:500). Subsequently, a secondary antibody reaction was carried out at room temperature (25°C) for 1 hour (Rabbit IgG HRP [manufactured by GE Healthcare] 1:5000).
  • an expression amount of CD63 present in an exosome in each blood serum was evaluated by Western blotting, using Purified Mouse Anti-Human CD63 (manufactured by Becton Dickinson Japan, clone: H5C6). The result is shown in FIG. 1 .
  • a signal of GPRC5C and a signal of CD63 present in an exosome in blood serum were detected as a concentration of GPRC5C and a concentration of CD63 present in the exosome, according to the method of Example 1.
  • the signal ratio of GPRC5C/CD63 is shown in FIG. 2 .
  • a concentration of GPRC5C present in an exosome in blood serum collected from a recurrent pancreatic cancer patient and a concentration of GPRC5C present in an exosome in blood serum collected from a healthy person were measured by the method of Example 1 using Western blotting, and both concentrations of GPRC5C were compared to each other.
  • a concentration of CD9 present in an exosome in each blood serum was measured by Western blotting, using a biotinized anti-CD9 antibody (1,000 times diluted) obtained by biotinizing an anti-CD9 antibody (8A12) (manufactured by Cosmo Bio Co., Ltd) and HRP-binding streptoavidin (manufactured by CST Corporation) (2,000 times diluted). From the measured concentration of GPRC5C and concentration of CD9, a ratio of the concentration of GPRC5C to the concentration of CD9 was calculated, and used as a signal ratio of GPRC5C/CD9. The signal ratio of GPRC5C/CD9 is shown in FIG. 4 .
  • the method and the reagent for examining a possibility of a subject having pancreatic cancer the method and the reagent for distinguishing pancreatic cancer from a pancreatic disease other than pancreatic cancer, the method and the reagent for monitoring a possibility of a subject having pancreatic cancer, a pancreatic cancer marker, a pancreatic cancer diagnosis reagent, and a method of determining biological sample of the present invention are useful for clinical diagnosis.

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Claims (22)

  1. Méthode in vitro d'examen d'une possibilité qu'un sujet soit atteint d'un cancer du pancréas comprenant :
    la mesure de GPRC5C (élément C du groupe 5 de la famille C des récepteurs couplés aux protéines G) présent dans un exosome dans un échantillon collecté du sujet.
  2. Méthode selon la revendication 1, comprenant les étapes suivantes :
    (1) une étape de mesure d'une concentration en GPRC5C présent dans l'exosome dans l'échantillon collecté du sujet ; et
    (2) une étape de détermination que la possibilité que le sujet soit atteint d'un cancer du pancréas est élevée dans un cas où la concentration en GPRC5C mesurée à l'étape (1) est supérieure à une concentration en GPRC5C présent dans un exosome dans un échantillon collecté d'un individu sain, et de détermination que la possibilité que le sujet soit atteint d'un cancer du pancréas est faible dans un cas où la concentration en GPRC5C mesurée à l'étape (1) est inférieure ou égale à la concentration en GPRC5C présent dans l'exosome dans l'échantillon collecté de l'individu sain.
  3. Méthode in vitro de différenciation d'un cancer du pancréas d'une maladie pancréatique autre qu'un cancer du pancréas chez un patient atteint d'une maladie pancréatique, comprenant :
    la mesure de GPRC5C (élément C du groupe 5 de la famille C des récepteurs couplés aux protéines G) présent dans un exosome dans un échantillon collecté d'un sujet atteint d'une maladie pancréatique.
  4. Méthode selon la revendication 3, comprenant les étapes suivantes :
    (1) une étape de mesure d'une concentration en GPRC5C présent dans l'exosome dans l'échantillon collecté d'un sujet atteint d'une maladie pancréatique; et
    (2) une étape de détermination qu'une possibilité que le sujet soit atteint d'un cancer du pancréas est élevée dans un cas où la concentration en GPRC5C mesurée à l'étape (1) est supérieure à une concentration en GPRC5C présent dans un exosome dans un échantillon collecté d'un patient atteint d'une maladie pancréatique autre que le cancer du pancréas, et de détermination que la possibilité que le sujet soit atteint d'une maladie pancréatique autre que le cancer du pancréas est élevée dans un cas où la concentration en GPRC5C mesurée à l'étape (1) est inférieure ou égale à la concentration en GPRC5C présent dans l'exosome dans l'échantillon collecté du patient atteint d'une maladie pancréatique autre qu'un cancer du pancréas.
  5. Méthode selon l'une quelconque des revendications 3 ou 4,
    dans laquelle la maladie pancréatique autre que le cancer du pancréas est une pancréatite.
  6. Méthode in vitro de surveillance d'une possibilité qu'un sujet soit atteint d'un cancer du pancréas, comprenant :
    la mesure de GPRC5C (élément C du groupe 5 de la famille C des récepteurs couplés aux protéines G) présent dans un exosome dans un échantillon collecté séquentiellement du sujet.
  7. Méthode selon la revendication 6, comprenant les étapes suivantes :
    (1) une étape de mesure d'une concentration en GPRC5C présent dans un exosome dans un échantillon séquentiellement collecté du sujet ; et
    (2) une étape de détermination que la possibilité que le sujet soit atteint d'un cancer du pancréas est élevée dans un cas où la concentration en GPRC5C mesurée à l'étape (1) est maintenue pour être supérieure à une concentration en GPRC5C présent dans un exosome dans un échantillon collecté d'un individu sain, et de détermination que la possibilité que le sujet soit atteint d'une maladie pancréatique est faible dans un cas où la concentration mesurée à l'étape (1) est maintenue pour être inférieure ou égale à la concentration en GPRC5C présent dans l'exosome dans l'échantillon collecté de l'individu sain.
  8. Méthode selon l'une quelconque des revendications 2, 4 ou 7,
    dans laquelle à l'étape (1), la mesure de la concentration en GPRC5C présent dans l'exosome dans l'échantillon collecté est réalisée en utilisant un exosome isolé de l'échantillon.
  9. Méthode selon l'une quelconque des revendications 2, 4, 7 ou 8,
    dans laquelle l'étape (1) est réalisée en utilisant une méthode de dosage immunologique.
  10. Méthode selon la revendication 9,
    dans laquelle la méthode de dosage immunologique est réalisée en utilisant un anticorps se liant à GPRC5C ou un fragment de liaison à GPRC5C de celui-ci.
  11. Méthode selon la revendication 9,
    dans laquelle la méthode de dosage immunologique est réalisée en utilisant l'anticorps se liant à GPRC5C ou un fragment de liaison à GPRC5C de celui-ci et un anticorps se liant à un antigène spécifiquement exprimé dans un exosome ou un fragment de liaison à un antigène de celui-ci, de préférence dans laquelle l'antigène spécifiquement exprimé dans un exosome est CD63, CD9, CD81, CD37, CD53, CD82, CD13, CD11, CD86, ICAM-1 (molécule d'adhésion intercellulaire-1), Rab5, l'annexine V, ou LAMP1 (protéine membranaire 1 associée aux lysosomes).
  12. Méthode selon l'une quelconque des revendications 1 à 11,
    dans laquelle l'échantillon est du sang.
  13. Réactif d'examen d'une possibilité qu'un sujet soit atteint d'un cancer du pancréas, comprenant :
    un réactif de mesure de GPRC5C (élément C du groupe 5 de la famille C des récepteurs couplés aux protéines G) présent dans un exosome dans un échantillon collecté d'un sujet, dans lequel le réactif est un réactif de dosage immunologique comportant un anticorps se liant à GPRC5C ou à un fragment de liaison à GPRC5C de celui-ci, et un anticorps se liant à un antigène spécifiquement exprimé dans un exosome ou un fragment de liaison à un antigène de celui-ci.
  14. Réactif de différenciation d'un patient atteint d'un cancer du pancréas d'un patient atteint d'une maladie pancréatique autre qu'un cancer du pancréas, de préférence une pancréatite, comprenant :
    un réactif de mesure de GPRC5C (élément C du groupe 5 de la famille C des récepteurs couplés aux protéines G) présent dans un exosome dans un échantillon collecté d'un patient atteint d'une maladie pancréatique, dans lequel le réactif est un réactif de dosage immunologique comportant un anticorps se liant à GPRC5C ou à un fragment de liaison à GPRC5C de celui-ci, et un anticorps se liant à un antigène spécifiquement exprimé dans un exosome ou un fragment de liaison à un antigène de celui-ci.
  15. Réactif de surveillance d'une possibilité qu'un sujet soit atteint d'un cancer du pancréas, comprenant :
    un réactif de mesure de GPRC5C (élément C du groupe 5 de la famille C des récepteurs couplés aux protéines G) présent dans un exosome dans un échantillon collecté séquentiellement du sujet, dans lequel le réactif est un réactif de dosage immunologique comportant un anticorps se liant à GPRC5C ou à un fragment de liaison à GPRC5C de celui-ci, et un anticorps se liant à un antigène spécifiquement exprimé dans un exosome ou un fragment de liaison à un antigène de celui-ci.
  16. Réactif selon l'une quelconque des revendications 13 à 15,
    dans lequel l'antigène spécifiquement exprimé dans un exosome est CD63, CD9, CD81, CD37, CD53, CD82, CD13, CD11, CD86, ICAM-1 (molécule d'adhésion intercellulaire-1), Rab5, l'annexine V, ou LAMP1 (protéine membranaire 1 associée aux lysosomes).
  17. Réactif selon l'une quelconque des revendications 13 à 16, comprenant en outre :
    un réactif d'isolement d'exosome.
  18. Réactif selon l'une quelconque des revendications 13 à 17,
    dans lequel l'échantillon est du sang.
  19. Utilisation in vitro d'un réactif comprenant GPRC5C (élément C du groupe 5 de la famille C des récepteurs couplés aux protéines G) ou un gène GPRC5C en tant que marqueur du cancer du pancréas.
  20. Réactif de diagnostic d'un cancer du pancréas, dans lequel le réactif est un réactif de dosage immunologique comportant un anticorps se liant à GPRC5C ou à un fragment de liaison à GPRC5C de celui-ci, et un anticorps se liant à un antigène spécifiquement exprimé dans un exosome ou un fragment de liaison à un antigène de celui-ci.
  21. Méthode in vitro de détermination d'un échantillon biologique comprenant :
    une étape de mesure d'une quantité de GPRC5C (élément C du groupe 5 de la famille C des récepteurs couplés aux protéines G) dans un échantillon biologique ; et
    une étape de détermination que l'échantillon biologique est un échantillon biologique d'un patient atteint d'un cancer du pancréas dans un cas où la quantité mesurée de GPRC5C est supérieure comparé à une quantité de GPRC5C dans un échantillon biologique d'un individu sain ou d'un patient atteint d'une maladie pancréatique autre qu'un cancer du pancréas, de préférence une pancréatite.
  22. Méthode in vitro de détermination d'un échantillon biologique comprenant :
    une étape de mesure d'une quantité d'expression d'un gène GPRC5C (élément C du groupe 5 de la famille C des récepteurs couplés aux protéines G) dans un échantillon biologique ; et
    une étape de détermination que l'échantillon biologique est un échantillon biologique d'un patient atteint d'un cancer du pancréas dans un cas où la quantité d'expression mesurée du gène est supérieure comparé à une quantité d'expression d'un gène GPRC5C dans un échantillon biologique d'un individu sain ou dans un échantillon biologique d'un patient atteint d'une maladie pancréatique autre qu'un cancer du pancréas, de préférence une pancréatite.
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Family Cites Families (10)

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Publication number Priority date Publication date Assignee Title
US20050260639A1 (en) * 2002-09-30 2005-11-24 Oncotherapy Science, Inc. Method for diagnosing pancreatic cancer
WO2005101001A2 (fr) 2004-04-13 2005-10-27 Bayer Healthcare Ag Methodes diagnostiques et therapeutiques pour lutter contre des maladies associees au recepteur couple a la proteine g, element c, groupe 5, famille c (gprc5c)
WO2005101012A1 (fr) * 2004-04-16 2005-10-27 Bayer Healthcare Ag Agents diagnostiques et therapeutiques pour des maladies associees au membre b, groupe 5, famille c de recepteur couple a une proteine g (gprc5b)
US8758991B2 (en) * 2006-04-26 2014-06-24 University Of Louisville Research Foundation, Inc. Isolation of membrane vesicles from biological fluids and methods of using same
US8617806B2 (en) * 2008-01-25 2013-12-31 Hansabiomed Ou Method to measure and characterize microvesicles in the human body fluids
KR101970908B1 (ko) * 2008-02-01 2019-04-19 더 제너럴 하스피탈 코포레이션 의학적 질환 및 병태의 진단, 예후, 및 치료에 있어서 미세소포체의 용도
CA2860144C (fr) 2011-12-22 2020-01-21 Takahiro Ochiya Procede d'analyse d'exosome, reactif pour l'utilisation dans l'analyse d'exosome, et appareil d'analyse d'exosome
CA2907606A1 (fr) 2013-04-08 2014-10-16 Shionogi & Co., Ltd. Methode de detection du cancer du colon
WO2016057629A1 (fr) * 2014-10-07 2016-04-14 Duke University Procédés et thérapeuthiques concernant la protéine r-spondine humaine et la protéine de récepteurs couplés aux protéines g contenant des motifs répétés riches en leucine
JP6611477B2 (ja) 2015-06-08 2019-11-27 キヤノン株式会社 撮像装置、発光制御方法、プログラム

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US20190331685A1 (en) 2019-10-31
JP6797427B2 (ja) 2020-12-09
EP3570032A1 (fr) 2019-11-20
US11965887B2 (en) 2024-04-23
CN110462404B (zh) 2023-06-02
JPWO2018131192A1 (ja) 2019-11-07
CN110462404A (zh) 2019-11-15
WO2018131192A1 (fr) 2018-07-19

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